WO2022112513A2

WO2022112513A2 - Nanoparticles comprising iron oxide particles embedded in polymeric micelles

Info

Publication number: WO2022112513A2
Application number: PCT/EP2021/083200
Authority: WO
Inventors: Natalie HORVAT; Martina U. MUCKENTHALER; Matthias W. Hentze; Tobias A. BAUER; Matthias BARZ
Original assignee: European Molecular Biology Laboratory; Universität Heidelberg
Priority date: 2020-11-27
Filing date: 2021-11-26
Publication date: 2022-06-02
Also published as: WO2022112513A3

Abstract

The present invention relates to a core-shell particle comprising a core cross-linked polymeric micelle (CCPM), and one or more iron oxide nanoparticles (IONs), wherein the one or more ION is located in the core of the CCPM. Further provided are methods for producing the core-shell particle, compositions comprising the same, the core-shell particle or the composition for use in medicine, and methods for modulating the activity of immune cells and methods of treating dysregulation of the immune system, cancer, anemia and nerve injuries.

Description

NANOPARTICLES COMPRISING IRON OXIDE PARTICLES EMBEDDED IN

POLYMERIC MICELLES

TECHNICAL FIELD OF THE INVENTION

The present invention relates to nanoparticles and in particular to iron oxide nanoparticles which are embedded in polymeric micelles, methods of their preparation, compositions comprising the nanoparticles and their use in medicine, in particular in modulating activity of immune cells and in treating dysregulation of the immune system, cancer and anemia.

BACKGROUND OF THE INVENTION

Iron is an essential cofactor for numerous cellular processes in the human body. Iron applied as iron-containing nanoparticle is internalized by macrophages, where metabolic changes are triggered. Superparamagnetic iron oxide nanoparticles (SPIONs) have been investigated for their potential as diagnostic and therapeutic systems, including the use as contrast agents for magnetic resonance imaging (MRI) (Weissleder et al, Am. J. Roentgenol., 1989, 152, 167-173; Jun et al., Angew. Chemie Int. Ed., 2008, 47, 5122-5135), magnetic hyperthermia (Perigoet al., Appl. Phys. Rev., 2015, 2, 041302), and magnetic drug targeting (MDT) (Baun and Bliimler, J. Magn. Magn. Mater., 2017, 439, 294-304; Tietze et al., Biochem. Biophys. Res. Commun., 2015, 468, 463-470; El-boubbou, Nanomedicine, 2018, 13, 929-952). Their use in these systems was mainly due to an exceptional biocompatibility in the body coupled to the natural way of processing iron through metabolic pathways (Recalcati et al, Eur. J. Immunol., 2010, 824-835). While these applications make use of the cooperative magnetic phenomena associated with magnetite or g-maghemite nanoparticles, ferumoxytol (Feraheme®, polyglucose-sorbitol-carboxy-methylether-coated SPIONs) is approved for an off-label use in the treatment of iron deficiency anaemia in patients with chronic kidney disease (Kowalczyket al, J. Nephrol., 2011, 24, 717-722) underlining the accessibility of the embedded nutrient iron. However, even though SPIONs have been developed and approved, some were withdrawn later due to immune-mediated toxicities (Foy and Labhasetwar, Biomaterials, 2011, 32, 9155-9158). The structural component that contributes to immunotoxicity is thought to be mainly the polymer coat, where certain chemical moieties can activate the complement system. It remains difficult to distinguish toxicities related to coating or particle instability where disintegration and aggregation can result in harm to metabolizing organs.

Certain diseases, such as cancer, atherosclerosis, traumatic nerve injury and autoimmune disorders are hallmarked by inflammation, whereby the infiltration of innate immune cells can exacerbate the disease condition (Costa da Silva et al, Front. Immunol., DOI:10.3389/fimmu.2017.0147915; Shenoy et al, Lab. Invest., 2017, 97, 494-49; Chinetti- Gbaguidi and Staels, Curr Opin Lipidol 2011, 22, 365-372). Large phagocytic cells, such as monocytes and monocyte derived macrophages, comprise a significant proportion of these infiltrating cells. Focus on these cells has garnered considerable interest particularly due to the phenomenon that macrophages undergo a phenotypic change known as polarization. A growing number of macrophage subtypes has been observed, both among tissue resident and peripheral patrolling cells, that are hallmarked by different functional capabilities, depending on niche derived stimuli, such as cytokines, chemokines and metabolites (Cairo et al., Trends Immunol., 2011, 32, 241-24757; Bao et al, Journals of Materials Chemistry 2018, 6(1280); Mebius and Kraal, 2005 Nat. Rev. Immunol., 8, 606-16 ). Recruited monocyte derived macrophages in diseased tissue, such as those residing in the periphery of solid cancers, are considered to mediate adaptive immunity, promote angiogenesis, tissue remodelling and repair, and often contribute to the aggressiveness of a cancer’s invasive front (Lewis and Pollard, Cancer Res., 2006, 66, 605-612). Apart from immune functions, macrophages play a central role in maintaining iron homeostasis, as they recycle hemoglobin-derived iron from senescent red blood cells (Recalcati et al, Eur. J. Immunol. 2010, 824-835; Sukhbaatar and Weichhart, Pharmaceuticals, 2018, 11, 137). The intricate connection between the immune function of macrophages and their role in iron metabolism was demonstrated by the exposure to metabolites such as free heme or iron that directly affect the macrophage activation state, leading not simply to changes in the expression of iron-regulatory genes but also in innate immune effector functions (Zanganeh et al., Nat. Nanotechnok, 2016, 11, 986-994; Costa da Silva et al, 2017; Shenoy et al, Lab. Invest., 2017, 97, 494-497).

By applying iron in the form of SPIONs within the tumour microenvironment, macrophages become activated, a process that correlates with inhibition of tumour growth in vivo (Zanganeh et al., Nat. Nanotechnok, 2016, 11, 986-994; Costa da Silva et al., 2017). SPIONs have demonstrated intrinsic therapeutic properties and recent studies suggest that the efficacy is due to release of iron from the particles’ core (Zanganeh et al. Nat. Nanotechnok 2016, 11, 986-994, Costa da Silva et ak, 2017). Since the degradation of the ION core is necessary to observe physiological effects, the composition of the polymer coating or shell is the main determinant of nanoparticle colloidal stability and timing of degradation, thus comprising the particles’ efficacy and pharmacokinetics profile (Cabral, et al., Nat. Nanotechnol., 2011, 6, 815-823; Talelli et al, Nano Today, 2015, 10, 93-117; Hareet al, Adv. Drug Deliv. Rev., 2017, 108, 25-38). The most common way to provide stable and prolonged circulation upon systemic administration is the coating of bare iron oxide nanoparticles with hydrophilic polymers. Among others, natural polymers such as dextran, chitosan, hyaluronic acid and starch, as well as synthetic polyvinyl alcohol, polyglutamate or polyethylene glycol have been previously used (Barrow et al, 2015, Chem. Soc. Rev., 6733- 6748; Arami et al, Chem. Soc. Rev., 2015, 44, 8576-8607; Mahmoudi et al, Adv. Drug Deliv. Rev., 2011, 63, 24-46).

There remains a need in the art for new forms of iron oxide nanoparticles (IONs), a need for new ways of administering IONs to achieve a long term iron supplementation and for therapies requiring administration of iron in general and for treating dysregulation of the immune system, cancer or anemia in particular.

SUMMARY OF THE INVENTION

The present invention has been accomplished in view of the above identified needs. In a first aspect, the invention provides a core-shell particle comprising a core cross-linked polymeric micelle (CCPM), and one or more iron oxide nanoparticles (IONs), wherein the one or more ION is located in the core of the CCPM.

According to one embodiment, the one or more IONs comprise Fe₂0₃ or Fe₃0₄ or a mixture thereof.

According to a preferred embodiment, the IONs have a diameter in the range of 5 to

20 nm.

According to a preferred embodiment, the IONs are paramagnetic, preferably superparamagnetic.

According to a preferred embodiment, the one or more ION is coated with a small molecule surfactant. Preferably, the small molecule surfactant is one or more fatty acid or monophosphoryl lipid. More preferably, the small molecule surfactant is one or more monounsaturated fatty acid. Most preferably, the small molecule surfactant is oleic acid.

According to one embodiment, the CCPM comprises a polymer comprising a thiol- reactive block consisting of between 1 and 1000 monomeric units of formula (C)

wherein n is 1 or 2;

R¹ is H, Ci-C4-alkyl, C2-C4-alkenyl or C2-C4-aklynyl;

R⁶ is independently selected from H, a group of formula (A), and a group of formula

(B)

wherein m is 1, 2, 3, 4, or 5;

R³ is independently selected from halogen, nitro, cyano and Ci-C4-alkanoyl; R⁴ is selected from R^a, Ci-Ci₆-alkyl, R^a-Ci-Ci₆-alkyl, C2-Ci6-alkenyl, and C2-

Ci₆-alkynyl, wherein the Ci-Ci₆-alkyl, C2-Ci6-alkenyl, and C2-Ci6-alkynyl are unsubstituted or carry 1, 2 or 3 substituents R^b, wherein R^b is independently selected from halogen, cyano, nitro, hydroxyl and thiol; wherein R^a is selected from

(i) phenyl;

(ii) 5- or 6-membered heteroaromatic monocyclic radicals having 1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N; (iii) 8- to 10-membered heteroaromatic bicarbocyclic radicals having

1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N; and (iv) 8- to 10-membered aromatic bicarbocyclic radicals, wherein said radicals (i) to (iv) are unsubstituted or carry 1, 2, 3 or 4 substituents R^c; wherein R^c is independently selected from the group consisting of halogen, cyano, nitro, hydroxyl, Ci-C4-alkyl, Ci-C4-alkoxy, C2-C4- alkenyl, C2-C4-aklynyl and Ci-C4-alkanoyl, wherein at least one monomeric unit of formula (C) R⁶ is a group of formula (A) or formula (B).

According to a preferred embodiment, each R³ is independently selected from the group consisting of fluoro, bromo and chloro, preferably wherein m is 5 and R³ is chloro, or m is 1, 2 or 3 and R³ is fluoro. Preferably or alternatively, R⁴ is selected from the group consisting of ethyl, butyl, isopropyl, hexyl and benzyl, wherein the benzyl is unsubstituted or carries 1, 2, 3 or 4 substituents R^c.

According to one embodiment, the core-shell particle further comprises at least one dye. The at least one dye is preferably conjugated to the amine group of the amphiphilic copolymer.

According to a further aspect, the present invention provides a composition comprising a plurality of the core-shell particles of the invention. Optionally, the composition further comprises a pharmaceutically acceptable carrier.

According to a further aspect, the present invention provides the core-shell particle of the invention or the composition of the invention for use in medicine.

According to a further aspect, the present invention provides the core-shell particle of the invention or the composition of the invention for use in immunotherapy or for use in treating dysregulation of the immune system, cancer or anemia.

According to yet another aspect, the present invention provides a method of producing an iron oxide nanoparticle-loaded core cross-linked polymeric micelle. The method comprises the steps of:

(a) combining iron oxide nanoparticles (IONs) with a polymer solution of reactive amphiphilic polysarcosine-block-poly(S-alkylsulfonyl cysteine) and/or reactive amphiphilic polysarcosine-block-poly(S-alkylsulfonyl homocysteine) in organic solvents, and allowing the polymers and the IONs to co-self-assemble in block selective solvents;

(b) core cross-linking the cysteine moieties of the polymers; and

(c) optionally conjugating a dye to the micelles.

According to one embodiment, step (a) further comprises dialyzing the solution comprising the IONs and the amphiphilic reactive block copolymers against organic solvents and subsequently against water. Additionally or alternatively, step (b) further comprises dialyzing the solution comprising the core cross-linked polymeric micelles against organic solvents and subsequently against water.

According to a preferred embodiment, the iron oxide nanoparticles in step (a) are coated with a small molecule surfactant. Preferably, the small molecule surfactant is one or more fatty acid or monophosphoryl lipid. More preferably, the small molecule surfactant is one or more monounsaturated fatty acid. Most preferably, the small molecule surfactant is oleic acid.

According to yet another embodiment, the CCPM comprises a plurality of amphiphilic polysarcosine-block-poly(S-alkylsulfonyl cysteine) copolymers and/or amphiphilic polysarcosine-block-poly(S-alkylsulfonyl homocysteine) copolymers.

According to a further aspect, the present invention provides an iron oxide nanoparticle-loaded core cross-linked polymeric micelle obtained by one of the methods of the invention.

According to yet another aspect, the present invention provides a method for modulating activity of immune cells. The method comprises administering the composition of the invention to one or more immune cells. Preferably, the one or more immune cells are macrophages. Preferably, activating macrophage activity comprises inducing a pro- inflammatory response in the macrophages or inducing macrophage polarization.

According to yet another aspect, the present invention provides a method for modulating dendritic cell activity. The method comprises administering the composition of the invention to one or more dendritic cells. Preferably, activating dendritic cell activity comprises inducing a pro-inflammatory response in the dendritic cells or inducing dendritic cell polarization.

According to yet another aspect, the present invention provides a method for modulating monocyte activity. The method comprises administering the composition of the invention to one or more monocytes. Preferably, activating monocyte activity comprises inducing a differentiation response in monocytes or inducing monocyte differentiation or maturation.

According to a further aspect, the present invention provides a method of treating dysregulation of the immune system in a patient in need thereof. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention. According to a further aspect, the present invention provides a method of treating cancer in a patient in need thereof. The cancer is preferably lung cancer. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention.

According to a further aspect, the present invention provides a method of treating anemia in a patient in need thereof. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention.

According to a further aspect, the present invention provides a method of treating a nerve injury in a patient in need thereof. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention.

According to a preferred embodiment, the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition of the invention.

Further embodiments and aspects of the invention will become apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the polymerization scheme of pSar_n-block-pCys(S02Et)_m.

Figure 2 schematically shows preparation of dye-labelled ION-loaded core cross- linked polymeric micelles (ION-CCPM^Cy5). Oleic acid-coated IONs (brown spheres) were loaded into polymeric micelles of reactive amphiphilic polypept(o)ide by co-self-assembly. Core cross-linking allows for chemoselective disulphide bond formation and anchoring to the iron oxide nanoparticle surface. Fluorescent dye Cy5 NHS-ester (star) was conjugated to the primary amine end group. Free dye was removed.

Figure 3 shows the particle characterization. (A) Image of ION-CCPM (left) and dye-labelled ION-CCPM⁰⁵'⁵ (right) dispersions in MilliQ water. (B) Image of the purification by extraction with dichloromethane (DCM) (left: first extraction, right: final extraction). (C) Single-angle dynamic light scattering of ION-Micelles and ION-CCPMS before and after lyophilization and redispersion. (D) For ION-CCPM⁰⁵'⁵, spherical morphologies and particle sizes below 100 nm were detected by atomic force microscopy. (E) Local clusters containing multiple iron oxide cores were detected by transmission electron microscopy of ION-CCPM⁰⁵'⁵.

Figure 4 shows Chemical Particle Analysis. (A) AT-IR spectroscopy indicates successful replacement of oleic acid-coating for IONs upon encapsulation in cross-linked ION-CCPMs. (B) Iron oxide content was determined from remaining weight as measured by thermogravimetric analysis in pure oxygen atmosphere. (C) HFIP-GPC indicates stable cross-linking and the absence of residual unconjugated dye or polymer for ION-CCPM^Cy5. (D) Neutral zeta-potentials were determined for both, ION-CCPM^Cy5 and CCPM '⁵, in 3 mM sodium chloride solution. (E) DLS of ION-CCPM^Cy5 measured at an angle of 30° indicates no aggregation in human blood serum accounting for colloidal stability.

Figure 5: ION-CCPMs^Cy5 and CCPMs '⁵ are efficiently taken up in macrophages. (A) BMDMs were treated with increasing concentrations of ION-CCPMs or CCPMs and internalization was measured by FACS fluorescence detection (Cy5). (B and C) Representative images of ION-CCPMs⁰⁵'⁵ or CCPMs⁰⁵'⁵ (red) taken up by BMDMs and the corresponding quantification of Cy5 fluorescence. Cells were incubated with 20 mM ION- CCPM°⁵'⁵ or CCPM°⁵'⁵ for 24 hours, then stained with Ibal (green), a cell surface marker for macrophages, and DAPI. Data reported as n ± SEM. One-way ANOVA (black) or students’ t-test (red): ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 6: ION-CCPMs^0y5 and CCPMs⁰⁵'⁵ are rapidly taken up in macrophages. BMDMs were treated with ION-CCPMs or CCPMs and internalization was measured by microscopy. Representative images of ION-CCPMs^0y5 or CCPMs⁰⁵'⁵ (red) taken up by BMDMs are. Cells were incubated with 20 pM ION-CCPM⁰⁵'⁵ or CCPM°⁵'⁵ for 1 hour, then stained with Ibal (green), a cell surface marker for macrophages, and DAPI.

Figure 7 shows that ION-CCPMs stimulate BMDMs. Cells were incubated with 20 pM ION-CCPMs, CCPMs, or ferric ammonium citrate (FAC). (A) Supernatant of cultures were used to measured lactate dehydrogenase (LDH) quantities at 490 nm after adding

CytoTox 96© substrate (Promega). Values are represented as a percentage of our 100% viable control at each time point. (B) After experimental incubation time, cells were incubated with CellTiter-Blue (Promega) for 4 hours and fluorescence was measured at 590 nm. Values are represented as fold change of 100% cell death value. Data reported as n ± SEM. n = 3 independent experiments. One-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, * indicates comparison to NT.

Figure 8 shows that ION-CCPMs release iron and induce ROS production in

BMDMs. (A) Transferrin receptor mRNA and protein detection in BMDM’s incubated with

20 pM ION-CCPMs, 20 pM CCPMs or 20 pM ferric ammonium citrate (FAC) for 6 or 24 hours, respectively. (B) Ferroportin mRNA expression after 6 hours. (C) Cytoplasmic or nuclear and mitochondrial ROS detection using CELLROX Orange and CELLROX Green probes, respectively, in BMDM’s after 4- and 18-hour incubation. Fluorescent intensities produced by ROS probes were measured by FACS. (D) Representative images of iron (blue) staining of BMDM’s incubated for 6 hours, 15 hours, or 24 hours with 20 mM ION-CCPMs, 20 pM CCPMs or 20 pM ferric ammonium citrate (FAC). Cells were stained with Peris’ Prussian blue and counterstained with nuclear fast red (pink). (E) Ferroportin mRNA expression after 18 hours. Data reported as n ± SEM. One-way ANOVA (black) or students’ t-test (red): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 9 shows that ION-CCPMs but not CCPM control induced inflammatory activation of macrophages. (A-B) Cells were incubated with 100 ng/mL LPS, 20 pM FAC, ION-CCPMs, CCPMs for 24 hours and cell surface protein expression was measured by FACS. (C) ILip, IL6, TNFa, or iNOS mRNA expression was measured in BMDMs treated with 100 ng/mL LPS, 20 pM ION-CCPMs, or 20 pM CCPMs for 6 hours. All values were normalized to the house keeping gene Rpll9 and represented as a fold change to the non- treated condition. Data reported as n ± SEM. One-way ANOVA (black) or students’ t-test (red): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 10 shows that ION-CCPMs and not CCPMs activate an inflammatory response in human macrophages. (A and B) Human peripheral monocytes were differentiated for 10 days using M-CSF to produce macrophages. Macrophages were incubated with 20 pM ION-CCPMs, Feraheme® (Amag Pharmaceuticals), CCPMs, or 100 ng/mL lipopolysaccharide (LPS). After 24 hours, cells were harvested for FACS analysis (A) or differential cytokine mRNA expression using qPCR (B). One-way ANOVA (black) or students’ t-test (red): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 11 shows CD86 and MHC II protein expression in macrophages following treatment with varying cysteine and homocysteine derivatives. Cells were incubated for 24 hours with 20 pM iron (ION-CCPMS, heme or FAC), CCPMs, L-cysteine (L-Cys), L- Cys(S0₂Et), L-Hcy(S0₂Et)), and cell surface markers CD86 and MHC II was measured using fluorescence dectection by FACS. Values are represented as normalized to control. One-way ANOVA (black) or students’ t-test (red): * p < 0.05, ** p < 0.01, *** p < 0.001,

**** p < 0_.0001_.

Figure 12 shows that ION-CCPMs induce sterile inflammation in macrophages. (A- D) BMDM’s were incubated with 100 ng/mL LPS, 20 pM FAC, 20 pM Heme, 20 pM ION- CCPMs or CCPMs for 18 hours. All values were normalized to the house keeping gene Rpll9 and represented as a fold change to the non-treated condition. mRNA expression of the indicated genes was quantified by qPCR using Sybergreen. Data reported as n ± SEM. One-way ANOVA (black) or student’s t-test (red): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < o.OOOl.

Figure 13 shows single-angle DLS of pSar-b-pCys(S0₂Et) block copolymers (PI PS) in DMSO.

Figure 14 shows the ¾ DOSY NMR spectrum of PI (pSar225-block-pCys(S02Et)33) in DMSO-de.

Figure 15 shows the 'H DOSY NMR spectrum of P2 (pSar₂oo-block-pCys(S0₂Et)i₇) in DMSO-de.

Figure 16 shows the ¾ DOSY NMR spectrum of P3 (pSari7o-block-pCys(S02Et)29) in DMSO-de.

Figure 17 shows particle degradation of ION-CCPMs in different concentrations of glutathione in carbonate buffer.

Figure 18 shows particle degradation of ION-CCPMs in different concentrations of glutathione in PBS.

Figure 19 shows non-heme iron content in the lungs and liver of mice treated with ION-CCPMs or PBS as control.

Figure 20 shows alterations in indicated hematological parameters measured in mice treated with ION-CCPMs or PBS as control.

Figure 21 shows flow cytometry results for innate immune cell populations of mice bronchoalveolar lavage (BAL) cells. Figure 21 A: accumulation of ION-CCPM fluorescence signals in different cell types over time in comparison to control cells treated with PBS. Figure 21B: numerical evaluation of the results shown in Fig. 21A.

Figure 22 shows changes in different cell surface markers on different types of macrophages in the lungs of mice upon administration of either ION-CCPMs or PBS as control.

Figure 23 shows time-dependent mRNA expression of pro-inflammatory cytokines 111/5, 116 and Tnfa, and of oxidative stress response proteins Ho-1 and Slc7al 1 in lung tissue treated either with ION-CCPMs or with PBS as control.

Figures 24 A to D show viability, rate of division and intracellular Lewis lung carcinoma (LLC) cell signal intensity in LLC cells co-cultured with bone marrow derived macrophages (BMDMs) after the addition of ION-CCPMs or CCPMs or non-treated (NT) (Figs. 24A-D). ION-CCPM signal compared to LLC (Fig. 24E), gene expression profile of Nqol (Fig. 24F) and NOS2 (Fig. 24G) in LLC cells and macrophages after co-culturing and ION-CCPM or CCPM treatment. Figure 25A shows evaluation of immune cell populations within lung tumors of mice treated with either ION-CCPMs, CCPMs, or PBS by flow cytometry. Figure 25B shows histological evaluation of iron content in lung tumors of mice treated with ION-CCPM (black arrows indicate tumor cells). Figure 25C shows number of tumors in lung cancer mice treated with ION-CCPMs compared to control mice treated with PBS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention shows that the novel iron sources provided can be used as adjuvant immunotherapeutic, preferably in combination with more traditional chemotherapeutic methods. Without wishing to be limited to any theory, compared to nanomedicine-based drug delivery, this approach relies on nanoparticle uptake in cells, particularly in macrophages, instead of deep tissue penetration, which circumvents an important barrier in active site targeting. The present invention shows that iron oxide nanoparticle-loaded core cross-linked polymeric micelles (ION-CCPMs) present a novel iron- containing formulation for immunomodulation of macrophages. The design concept merges steric shielding and core cross-linking with the introduction of anchor groups to grant colloidal stability and stimuli-responsive degradation. The present invention further shows that ION-CCPMs have high colloidal stability in human blood plasma and potently induce an immunomodulatory effect on macrophages in vitro. Without wishing to be bound by any theory, the ION-CCPMs of the present invention can be stimuli-responsive, meaning that they can degrade in response to a trigger. Preferably, such trigger is glutathione.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and cell biology are employed which are explained in the literature in the field.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise.

The term "pharmaceutically acceptable carrier", as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, surfactant, stabilizer, physiological buffer solution or vehicle with which the nanoparticles are administered. Pharmaceutical carriers are also called pharmaceutical excipients and can have a liquid, solid or gel-like texture. Liquid carriers include but are not limited to sterile liquids, such as saline solutions in water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. In a preferred embodiment of the invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical excipients comprise starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed e.g. in the U.S. Pharmacopeia, or other national or multinational regulatory authorities, or generally recognized pharmacopeia for use in animals, and more particularly in humans.

In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. The present invention shows for the first time that iron oxide nanoparticles (IONs) can be formulated into a core-shell particle comprising a core cross-linked polymeric micelle (CCPM), wherein the one or more ION is located in the core of the CCPM. Preferably, a content of iron oxide of between around 20 and 90 weight-% compared to the entire weight of the ION-CCPMs can be loaded in the ION-CCPMs of the present invention, more preferably around 30, 40, 50, 60, 70 and 80 weight-%, most preferably around 70 weight-%. If seen from a particulate level, a number of between around 1 and 100 iron oxide nanoparticles can be preferably present in each ION-CCPM of the present invention, more preferably between around 10 and 90, between around 20 and 80, between around 30 and 70, between around 40 and 60 iron oxide nanoparticles. Particularly preferred is a range of between around 1 and 25 iron oxide nanoparticles in each ION-CCPM, of between around 2 and 20, of between around 3 and 15, and most preferably of between 4 and 6 iron oxide nanoparticles in each ION- CCPM.

When preparing a composition for administration, such as a pharmaceutical composition according to the invention, the ION-CCPMs are preferably dispersed in a suitable carrier fluid selected from the group consisting of but not limited to 0.9 % saline, PBS or the like. The ION-CCPMs are preferably present in a total mass concentration of between about 0.1 and 500 g/L, more preferably between about 1 and 400 g/L, between about 2 and 300 g/L, between about 3 and 200 g/L, between about 4 and 100 g/L, and most preferably between about 5 and 50 g/L.

The iron concentration is preferably between about 0.1 and 1000 mmol/L, more preferably between about 0.5 and 900 mmol/L, between about 1.0 and 800 mmol/L, between about 2 and 700 mmol/L, between about 5 and 600 mmol/L, and most preferably between about 10 and 500 mmol/L.

According to one embodiment of the present invention, the one or more IONs comprise Fe₂0₃ or Fe₃0₄ or a mixture thereof. Iron oxide nanoparticles usually have a diameter of between 1 and 100 nm. The IONs used in the present invention preferably have a diameter in the range of 1 to 50 nm, 2 to 40 nm, and more preferably in the range of 5 to 20 nm, such as in the range of 6 to 19 nm, 7 to 18 nm, 8 to 17 nm, 9 to 16 nm, 10 to 15 nm, 11 to 14 nm, or 12 to 13 nm. Particularly preferred is an average diameter of the IONs of around 6 nm.

The entire core-shell particle preferably has an overall average diameter of around between 50 to 150 nm, between 60 to 120 nm, between 70 to 100 nm. Preferably, the overall average diameter of the core-shell particle of the present invention is about 80 nm. Preferred albeit not necessary for putting the invention into practice are paramagnetic and superparamagnetic iron oxide particles. In a most preferred embodiment, the IONs are superparamagnetic iron oxide particles (SPIONs).

It is preferred to coat the one or more ION with a small molecule surfactant. Preferably, the small molecule surfactant is one or more fatty acid or monophosphoryl lipid. More preferably, the small molecule surfactant is one or more monounsaturated fatty acid (MUFA). Preferred monounsaturated fatty acids are preferably selected from the group consisting of oleic acid, elaidic acid, vaccenic acid, paullinic acid, palmitoleic acid, gondoic acid, erucic acid, nervonic acid, myristoleic acid, sapienic acid, eicosenoic acid, crotonic acid and combinations thereof. Most preferably, the small molecule surfactant is oleic acid.

For the preparation of functional core cross-linked polymeric micelles (CCPMs), preferably polysarcosine-block-poly(S-alkylsulfonyl cysteine) block copolypept(o)ides are used as disclosed in EP 2 942 348. The polysarcosine-block-poly(S-alkylsulfonyl cysteine) block copolypept(o)ides have the advantage of conferring more stability to the ION- containing micelles, thereby preventing disintegration or aggregation upon injection into the bloodstream.

Polypept(o)ides combine the shielding properties of the polypeptoide polysarcosine (pSar, poly(N-methyl glycine)) with the multi-functionality of polypeptides. Synthesis can be conveniently done by living nucleophilic ring opening polymerization of the respective amino acid N-carboxy anhydrides. For the formation of stimuli-responsive CCPMs, pSar-b-pCys (SO2R) co-polymers uniquely offer secondary structure-directed self-assembly into either spherical or worm-like micelles. In a second step, the reactive S-alkysulfonyl group enables chemo-selective formation of asymmetric disulphides upon reaction with dithiols. Disulphide- stabilized-nanoparticles are stable in circulation while demonstrating compartment specific degradation profiles, such as when internalized by cells. This technique thus allows for a precisely tailored core polarity and function independent from the selected morphology.

Hence, according to one embodiment, the CCPM comprises a polymer comprising a thiol-reactive block consisting of between 1 and 1000 monomeric units of formula (C)

wherein n is 1 or 2;

R¹ is H, Ci-C4-alkyl, C2-C4-alkenyl or C2-C4-aklynyl;

(B)

wherein m is 1, 2, 3, 4, or 5;

(i) phenyl;

According to a preferred embodiment, each R³ is independently selected from the group consisting of fluoro, bromo and chloro, preferably wherein m is 5 and R³ is chloro, or m is 1, 2 or 3 and R³ is fluoro. Preferably or alternatively, R⁴ is selected from the group consisting of ethyl, butyl, hexyl and benzyl, wherein the benzyl is unsubstituted or carries 1, 2, 3 or 4 substituents R^c. Particularly preferred is ethyl.

Without wishing to be bound by any theory, the dense polysarcosine corona prevents aggregation and grants colloidal stability, while disulfide bonds in the core compartment made from cysteine and the multifunctional thiol such as lipoic acid enable stimuli-responsive nanoparticle degradation. Other cross-linkers can also be used in the present invention. These include but are not limited to lipoic acid, dihydro-lipoic acid, azidopropyl-liponamide or a peptide based cross-linker such as peptides with sequences of cysteine and sarcosine or homocysteine and sarcosine amino acids. Trifunctional cross-linkers consisting of alternating cysteine/sarcosine or homocysteine/sarcosine amino acids, such as: Cys-Sar-Cys-Sar-Cys or Hcy-Sar-Hcy-Sar-Hcy are particularly preferred. Other peptides with a distinct biologic background and function can alternatively be used, e.g. hepcidin. Most preferably, the multifunctional thiol is lipoic acid.

For better visualization and detection, the core-shell particle may further comprises at least one dye. Such dye is preferably conjugated to the amine group of the amphiphilic copolymer but can alternatively also be conjugated to any other suitable part or structure of the copolymer.

A composition according to the present invention comprises a plurality of the core shell particles of the invention. Optionally, the composition further comprises one or more selected from pharmaceutically acceptable carriers and pharmaceutically acceptable excipients, such as at least two, at least three, or at least four or more pharmaceutically acceptable carriers. Such composition is herein also referred to as pharmaceutical composition. In a preferred embodiment of the invention, the pharmaceutical composition is customized for the treatment of a disease or disorder. As used herein, “treat”, “treating” or “treatment” of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s); (f) reduction of mortality after occurrence of a disease or a disorder; (g) healing; and (h) prophylaxis of a disease. The term “ameliorating” is also encompassed by the term “treating”. As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis” of a disease or disorder means preventing that such disease or disorder occurs in patient.

In a particularly preferred embodiment of the invention, a treatment with a pharmaceutical composition according to the invention comprises the treatment of an individual in need of such treatment.

The pharmaceutical composition contemplated by the present invention may be formulated in various ways well known to one of skill in the art. For example, the pharmaceutical composition of the present invention may be in liquid form such as in the form of solutions, emulsions, or suspensions. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, preferably for intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intrap eritoneal intracoronary, intra-cardiac administration, or administration via mucous membranes, preferably for intravenous, subcutaneous, or intraperitoneal administration. A preparation for oral or anal administration is also possible. Preferably, the pharmaceutical composition of the present invention is in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9, more preferably to a pH of from 5 to 7), if necessary. The pharmaceutical composition is preferably in unit dosage form. In such form the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of pharmaceutical composition such as vials or ampoules.

The pharmaceutical composition is preferably administered through the intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary or intra-cardiac route, wherein other routes of administration known in the art are also comprised. The pharmaceutical composition is preferably administered through one or more bolus injection(s) and/or infusion(s), preferably in a pharmaceutically accepted carrier. A most preferred route of administration is via inhalation.

If the pharmaceutical composition is used as a treatment for an individual, the use of the pharmaceutical composition can replace the standard treatment for the respective disease or condition or can be administered additionally to the standard treatment. In the case of an additional use of the pharmaceutical composition, the pharmaceutical composition can be administered before, simultaneously or after a standard therapy.

It is further preferred that the pharmaceutical composition is administered once or more than once. This comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 times. The time span for the administration of the pharmaceutical is not limited. Preferably, the administration does not exceed 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 weeks. Most preferably, the administration does not exceed eight weeks.

A single dose of the pharmaceutical composition, can independently form the overall amount of administered doses, or the respective time span of administration can include administration as one or more bolus injection(s) and/or infusion(s).

According to one embodiment, the active ingredient is administered to a cell, a tissue or an individual in an effective amount. An “effective amount” is an amount of an active ingredient sufficient to achieve the intended purpose. The active ingredient in the composition of the present invention is the core-shell particle of the present invention, either alone or in combination with other suitable active ingredients, such as other therapeutic agents. The effective amount of a given active ingredient will vary with parameters such as the nature of the ingredient, the route of administration, the size and species of the individual to receive the active ingredient, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art. As used in the context of the invention, "administering" includes in vivo administration to an individual as well as administration directly to cells or tissue in vitro or ex vivo.

The core-shell particle of the invention and the composition of the invention are particularly suitable for use in medicine. Particularly preferred uses include the core-shell particle of the invention or the composition of the invention for use in treating, preventing or ameliorating dysregulation of the immune system. A dysregulation of the immune system or immune dysregulation is any proposed or confirmed breakdown or maladaptive change in molecular control of immune system processes. Preferably, the immune dysregulation is or is caused by inflammation, preferably autoinflammatory diseases, autoimmune diseases, dysregulation of lymphocyte homeostasis, hypersensitivity reactions, immune dysregulation polyendocrine opathyenteropathy X-linked syndrome (IPEX), autoimmune polyendocrinopathy candidiasis-endodermal dystrophy (APECED), Omenn syndrome, Wiskott-Aldrich syndrome, a T cell immunodeficiency, immune dysregulation associated with stress, preferably leading to chronic inflammation, aging of the immune system, dysregulation caused by or in response to substances such as toxins. Treating, preventing or ameliorating dysregulation of the immune system also includes initiating an immune dysregulation in such cases in which a naturally occurring innate immune system supports e.g. tumor growth. The ION-CCPM nanoparticles of the present invention then usher a “dysregulation” in order to initiate anti-tumor effects.

Particularly preferred uses also include the core-shell particle of the invention or the composition of the invention for use in immunotherapy.

Particularly preferred uses also include the core-shell particle of the invention or the composition of the invention for use in treating, preventing or ameliorating cancer. The cancer may be any cancer, preferably selected from the group consisting of lymphocytic cancer, myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer such as gastrointestinal carcinoid tumor, gastric cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum cancer, omentum cancer, mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer. A preferred cancer is lung, colorectal, melanoma cancer or cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. According to a preferred embodiment, the cancer is lung cancer.

Particularly preferred uses also include the core-shell particle of the invention or the composition of the invention for use in treating, preventing or ameliorating anemia. Anemia is commonly understood as a decrease in the total amount of red blood cells (RBCs) or hemoglobin in the blood, or a lowered ability of the blood to carry oxygen. A preferred form of anemia is selected from the group consisting of pure red cell aplasia, aplastic anemia, Fanconi anemia, anemia of kidney failure, anemia of endocrine disorders, disturbance of proliferation and maturation of erythroblasts, pernicious anemia, non-pernicious megaloblastic anemia, anemia of folate deficiency, megaloblastic anemia, anemia of prematurity, iron deficiency anemia, Thalassemias, congenital dyserythropoietic anemia, anemia of kidney failure, myelophthisic anemia or myelophthisis, myelodysplastic syndrome, anemia of chronic inflammation, and leukoerythroblastic anemia. Preferably, the anemia is iron deficiency anemia.

Particularly preferred uses also include the core-shell particle of the invention or the composition of the invention for use in treating, preventing or ameliorating nerve injuries. A nerve injury is an injury to nervous tissue such as the spinal cord, and includes neurapraxia, axonotmesis and neurotmesis. A preferred nerve injury is spinal cord injury.

According to a further aspect of the present invention, provided is a method of producing the iron oxide nanoparticle-loaded core cross-linked polymeric micelle of the present invention. A schematic illustration of the process is illustrated in Figure 2. The method of producing the iron oxide nanoparticle-loaded core cross-linked polymeric micelle of the present invention comprises the steps of:

(b) core cross-linking the cysteine moieties of the polymers; and

(c) optionally conjugating a dye to the micelles.

The organic solvent in step (a) is preferably selected from the group consisting of chloroform, dimethyl sulfoxide, N,N-dimethyl formamide, N,N-diethyl acetamide, and any combination thereof. More preferably, the organic solvent in step (a) is chloroform, dimethyl sulfoxide or a combination thereof.

The block selective solvent in step (a) is preferably water since the polycysteine block is not soluble in water, while polysarcosine is very well soluble in water. The polycysteine block thus assembles and forms the core embedding the surfactant-coated iron oxide nanoparticles, which are also insoluble in water. Based on this description, the skilled person easily recognizes further alternative block selective solvents. Preferably, the core-cross-linking in step (b) is performed by using a multifunctional thiol. This multifunctional thiol is preferably selected from the group consisting of but not limited to lipoic acid, dihydro-lipoic acid, azidopropyl-liponamide or a peptide based cross linker such as peptides with sequences of cysteine and sarcosine or homocysteine and sarcosine amino acids. Trifunctional cross-linkers consisting of alternating cysteine/sarcosine or homocysteine/sarcosine amino acids, such as: Cys-Sar-Cys-Sar-Cys or Hcy-Sar-Hcy-Sar- Hcy are particularly preferred. Other peptides with a distinct biologic background and function can alternatively be used, e.g. hepcidin. Most preferably, the multifunctional thiol is lipoic acid.

According to one embodiment, step (a) further comprises dialyzing the solution comprising the IONs and the amphiphilic reactive block copolymers against organic solvents, preferably chloroform or dimethyl sulfoxide, and subsequently against water. Additionally or alternatively, step (b) further comprises dialyzing the solution comprising the core cross- linked polymeric micelles against organic solvents and subsequently against water.

According to a preferred embodiment, the iron oxide nanoparticles in step (a) are coated with a small molecule surfactant. Preferably, the small molecule surfactant is one or more fatty acid or monophosphoryl lipid. More preferably, the small molecule surfactant is one or more monounsaturated fatty acid (MUFA). Preferred monounsaturated fatty acids are preferably selected from the group consisting of oleic acid, elaidic acid, vaccenic acid, paullinic acid, palmitoleic acid, gondoic acid, erucic acid, nervonic acid, myristoleic acid, sapienic acid, eicosenoic acid, crotonic acid and combinations thereof. Most preferably, the small molecule surfactant is oleic acid.

According to yet another embodiment, the CCPM comprises a plurality of amphiphilic polysarcosine-block-poly(S-alkylsulfonyl cysteine) copolymers and/or amphiphilic polysarcosine-block-poly(S-alkylsulfonyl homocysteine) copolymers. The copolymers are preferably reacted with thiol-based cross-linkers. The thiol-based cross linker is preferably selected from the group consisting of lipoic acid, dihydro-lipoic acid, azidopropyl-liponamide or a peptide based cross-linker such as peptides with sequences of cysteine and sarcosine or homocysteine and sarcosine amino acids. Trifunctional cross-linkers consisting of alternating cysteine/sarcosine or homocysteine/sarcosine amino acids, such as: Cys-Sar-Cys-Sar-Cys or Hcy-Sar-Hcy-Sar-Hcy are particularly preferred. Other peptides with a distinct biologic background and function can alternatively be used, e.g. hepcidin. Most preferably, the multifunctional thiol is lipoic acid.

According to a preferred embodiment, oleic acid-coated IONs are loaded into polymeric micelles of reactive amphiphilic polysarcosine-block-poly(S-ethylsulfonyl) cysteine by co-self-assembly in chloroform/DMSO mixtures and dialysis against water. Core cross-linking with a-dihydro lipoic acid allows for chemoselective disulphide bond formation and anchoring to the iron oxide nanoparticle surface. Fluorescent dye Cy5 NHS- ester can be conjugated to the primary amine end group. Free dye is then removed, preferably by repetitive extraction with dichloromethane followed by spin-filtration. Azide end groups on the outer particle shell generally permit the introduction of ligands using click chemistry.

The present invention also relates to and provides an iron oxide nanoparticle-loaded core cross-linked polymeric micelle (ION-CCPM) obtained by one of the methods of the invention. Such ION-CCPM preferably contains polysarcosine-block-polycysteine copolymers which are cross-linked with thiol-carrying cross-linkers.

The present invention also provides a method for modulating activity of one or more immune cells. Immune cells can be selected from the group consisting of phagocytes, lymphocytes, granulocytes, lymphoid cells, monocytes, leukocytes, dendritic cells, macrophages and combinations thereof. Preferably, dendritic cells, macrophages and/or monocytes are activated. The method comprises administering the composition of the invention to the one or more cells. The methods disclosed herein are preferably in vitro methods.

Therefore, according to a preferred embodiment, the present invention provides a method for modulating macrophage activity. The method comprises administering the composition of the invention to one or more macrophages. Preferably, activating macrophage activity comprises inducing a pro-inflammatory response in the macrophage or inducing macrophage polarization.

According to a preferred embodiment, the present invention provides a method for modulating dendritic cell activity. The method comprises administering the composition of the invention to one or more dendritic cell. Preferably, activating dendritic cell activity comprises inducing a pro-inflammatory response in the dendritic cells or inducing dendritic cell polarization.

According to a preferred embodiment, the present invention provides a method for modulating monocyte activity. The method comprises administering the composition of the invention to one or more monocytes. Preferably, activating monocyte activity comprises inducing a differentiation response in monocytes or inducing monocyte differentiation or maturation. The present invention also provides a method of treating, preventing or ameliorating dysregulation of the immune system in a patient in need thereof. The method comprises the step of administering an effective amount of the composition of the invention to the patient in need thereof. A dysregulation of the immune system or immune dysregulation is any proposed or confirmed breakdown or maladaptive change in molecular control of immune system processes. Preferably, the immune dysregulation is or is caused by inflammation, preferably autoinflammatory diseases, autoimmune diseases, dysregulation of lymphocyte homeostasis, hypersensitivity reactions, immune dysregulation polyendocrine opathyenteropathy X-linked syndrome (IPEX), autoimmune polyendocrinopathy candidiasis- endodermal dystrophy (APECED), Omenn syndrome, Wiskott-Aldrich syndrome, a T cell immunodeficiency, immune dysregulation associated with stress, preferably leading to chronic inflammation, aging of the immune system, dysregulation caused by or in response to substances such as toxins. Treating, preventing or ameliorating dysregulation of the immune system also includes initiating an immune dysregulation in such cases in which a naturally occurring innate immune system supports e.g. tumour growth. The ION-CCPM nanoparticles of the present invention then usher a “dysregulation” in order to initiate anti-tumor effects.

The present invention also provides a method of treating cancer in a patient in need thereof. The method comprises the step of administering an effective amount of the composition of the invention to the patient in need thereof. The cancer may be any cancer, preferably selected from the group consisting of but not limited to lymphocytic cancer, myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer such as gastrointestinal carcinoid tumor, gastric cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum cancer, omentum cancer, mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer. A preferred cancer is lung, colorectal, melanoma cancer and cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis.

The present invention also provides a method of treating anemia in a patient in need thereof. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention. The anemia is preferably selected from the group consisting of but not limited to pure red cell aplasia, aplastic anemia, Fanconi anemia, anemia of kidney failure, anemia of endocrine disorders, disturbance of proliferation and maturation of erythroblasts, pernicious anemia, non-pemicious megaloblastic anemia, anemia of folate deficiency, megaloblastic anemia, anemia of prematurity, iron deficiency anemia, Thalassemias, congenital dyserythropoietic anemia, anemia of kidney failure, myelophthisic anemia or myelophthisis, myelodysplastic syndrome, anemia of chronic inflammation, and leukoerythroblastic anemia. Preferably, the anemia is iron deficiency anemia.

The present invention also provides a method of treating a nerve injury in a patient in need thereof. The method comprises the step of administering to the patient in need thereof an effective amount of the composition of the invention. The nerve injury is selected form the group consisting of but not limited to neurapraxia, axonotmesis and neurotmesis. A preferred form of nerve injury is spinal cord injury.

The composition is preferably administered through the intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, intracoronary or intra-cardiac route, wherein other routes of administration known in the art are also comprised. The pharmaceutical composition is preferably administered through one or more bolus injection(s) and/or infusion(s), preferably in a pharmaceutically accepted carrier. A most preferred route of administration is via inhalation.

The active ingredient is thereby administered to a cell, a tissue or an individual in an effective amount. An “effective amount” is an amount of an active ingredient sufficient to achieve the intended purpose. The active ingredient in the composition of the present invention is the core-shell particle of the present invention, either alone or in combination with other suitable active ingredients such as other therapeutic agents. The effective amount of a given active ingredient will vary with parameters such as the nature of the ingredient, the route of administration, the size and species of the individual to receive the active ingredient, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art. As used in the context of the invention, "administering" includes in vivo administration to an individual as well as administration directly to cells or tissue in vitro or ex vivo.

The present inventors developed an iron-containing formulation that displays colloidal stability but allows for stimuli-responsive degradation and iron release. Iron oxide nanoparticles have been embedded into polymeric micelles of polysarcosine-block poly(S- ethylsulfonyl-L-cysteine) copolymers. These micelles have been further cross-linked, resulting in ION-loaded core cross-linked polymeric micelles (ION-CCPMs).

As is shown in the following examples, when tested in cells, it was found that the ION-CCPMs according to the invention are preferentially taken up by bone marrow-derived macrophages (BMDMs) compared to e.g. primary murine hepatocytes or cancer cells. Moreover, the catabolism of ION-CCPMs modulates macrophage activity in a time- and dose-dependent manner. In comparison to the shell only (CCPMs), ION-CCPMs induce a strong pro-inflammatory response, whereby the expression of pro-inflammatory surface markers (CD86, CD80, CD38) and cytokines (TNFa, iNOS, PAb) is strongly increased. ION- CCPMs are taken up within one hour and metabolized in as little as 4 hours. Cells initially store ION-CCPMs and catabolize these nanoparticles within at least 120 hours, without overwhelming the system. ION-CCPMs are thus biocompatible and particularly useful for the treatment of diseases where dysregulation of the innate immune system occurs.

The present invention further provides a novel method for nanoparticle synthesis via a self-assembly process, allowing for uniform and replicable development. The protocol demonstrates the potential for future drug development in scaled up industry standards.

In particular, the present invention pertains to the following items:

Item 1 : A core-shell particle comprising

(i) a core cross-linked polymeric micelle (CCPM), and

(ii) one or more iron oxide nanoparticles (IONs), wherein the one or more ION is located in the core of the CCPM.

Item 2: The core-shell particle of item 1, wherein the one or more IONs comprise Fe₂0₃ or Fe₃0₄ or a mixture thereof; and/or wherein the IONs have a diameter in the range of 5 to 20 nm.

Item 3 : The core-shell particle of item I or 2, wherein the IONs are paramagnetic, preferably superparamagnetic.

Item 4: The core-shell particle of any one of items I to 3, wherein the one or more ION is coated with a small molecule surfactant, preferably wherein the small molecule surfactant is one or more fatty acid or monophosphoryl lipid, more preferably wherein the small molecule surfactant is one or more monounsaturated fatty acid, most preferably wherein the small molecule surfactant is oleic acid.

Item 5 : The core-shell particle of any one of items 1 to 4, wherein the CCPM comprises a polymer comprising a thiol-reactive block consisting of between 1 and 1000 monomeric units of formula (C)

wherein n is 1 or 2;

R¹ is H, Ci-C4-alkyl, C2-C4-alkenyl or C2-C4-aklynyl;

(B)

wherein m is 1, 2, 3, 4, or 5;

(i) phenyl;

(ii) 5- or 6-membered heteroaromatic monocyclic radicals having 1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N; (iii) 8- to 10-membered heteroaromatic bicarbocyclic radicals having 1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N; and

(iv) 8- to 10-membered aromatic bicarbocyclic radicals, wherein said radicals (i) to (iv) are unsubstituted or carry 1, 2, 3 or 4 substituents R^c; wherein R^c is independently selected from the group consisting of halogen, cyano, nitro, hydroxyl, Ci-C4-alkyl, Ci-C4-alkoxy, C2-C4- alkenyl, C2-C4-aklynyl and Ci-C4-alkanoyl, wherein at least one monomeric unit of formula (C) R⁶ is a group of formula (A) or formula (B).

Item 6: The core-shell particle of item 5, wherein each R³ is independently selected from the group consisting of fluoro, bromo and chloro, preferably wherein m is 5 and R³ is chloro, or m is 1, 2 or 3 and R³ is fluoro, and/or

R⁴ is selected from the group consisting of ethyl, butyl, isopropyl, hexyl and benzyl, wherein the benzyl is unsubstituted or carries 1, 2, 3 or 4 substituents R^c.

Item 7 : The core-shell particle of any one of items 1 to 6, further comprising at least one dye, wherein the at least one dye is preferably conjugated to the amine group of the amphiphilic copolymer.

Item 8: A composition comprising a plurality of the core-shell particles according to any one of claims 1 to 7, optionally, further comprising a pharmaceutically acceptable carrier.

Item 9: The core-shell particle of any of items 1 to 7 or the composition of item 8 for use in medicine.

Item 10: The core-shell particle of any of items 1 to 7 or the composition of item 8 for use in immunotherapy or for use in treating dysregulation of the immune system, cancer or anemia.

Item 11: A method of producing an iron oxide nanoparticle-loaded core cross-linked polymeric micelle, the method comprises the steps of:

(b) core cross-linking the cysteine moieties of the polymers; and

(c) optionally conjugating a dye to the micelles. Item 12: The method according to item 11, wherein step (a) further comprises dialyzing the solution comprising the IONs and the amphiphilic reactive block copolymers against organic solvents and subsequently against water; and/or wherein step (b) further comprises dialyzing the solution comprising the core cross- linked polymeric micelles against organic solvents and subsequently against water.

Item 13: The method according to item 11 or 12, wherein the iron oxide nanoparticles in step (a) are coated with a small molecule surfactant, preferably wherein the small molecule surfactant is one or more fatty acid or monophosphoryl lipid, more preferably wherein the small molecule surfactant is one or more monounsaturated fatty acid, most preferably wherein the small molecule surfactant is oleic acid.

Item 14: The method according to any one of items 10 to 13, wherein the CCPM comprises a plurality of amphiphilic polysarcosine-block-poly(S-alkylsulfonyl cysteine) copolymers and/or amphiphilic polysarcosine-block-poly(S-alkylsulfonyl homocysteine) copolymers, which are reacted with thiol-based cross-linkers.

Item 15: An iron oxide nanoparticle-loaded core cross-linked polymeric micelle obtained by the method of any one of items 11 to 14.

Item 16: A method for modulating activity of immune cells, comprising administering the composition according to item 8 to one or more immune cells, preferably wherein the immune cell is a macrophage, more preferably wherein activating the activity of the macrophage comprises inducing a pro-inflammatory response in the macrophage or inducing macrophage polarization.

Item 17: A method for modulating dendritic cell activity, comprising administering the composition according to item 8 to one or more dendritic cells, preferably wherein activating dendritic cell activity comprises inducing a pro-inflammatory response in the one or more dendritic cells or inducing dendritic cell polarization.

Item 18: A method for modulating monocyte activity, comprising administering the composition according to item 8 to one or more monocytes, preferably wherein activating monocyte activity comprises inducing a differentiation response in monocytes or inducing monocyte differentiation or maturation.

Item 19: A method of treating dysregulation of the immune system in a patient in need thereof, the method comprising administering an effective amount of the composition according to item 8 to the patient in need thereof. Item 20: A method of treating cancer in a patient in need thereof, the method comprising administering an effective amount of the composition according to item 8 to the patient in need thereof.

Item 21: A method of treating anemia in a patient in need thereof, the method comprising administering an effective amount of the composition according to item 8 to the patient in need thereof.

Item 22: A method of treating a nerve injury in a patient in need thereof, the method comprising administering an effective amount of the composition according to item 8 to the patient in need thereof.

Item 23: The method according to any one of items 19 to 22, wherein the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition.

EXAMPLES

The Examples are designed to further illustrate the present invention and to serve a better understanding. They are not to be construed as limiting the scope of the invention in any way.

Materials

Unless stated otherwise, solvents were purchased from Sigma Aldrich. THF and n- hexane were dried over Na and freshly distilled prior to use. DMF was bought from Acros (99.8 %, Extra Dry over Molecular Sieve), freeze-pumped prior to use to remove residual dimethylamine, and handled in the absence of light. HFIP was purchased from Fluorochem, deuterated solvents from Deutero and were used as received. MilliQ water was prepared using a MILLI-Q® Reference A+ System. Water was used at a resistivity of 18.2 MW-cm ¹ and total organic carbon of <5 ppm. Diphosgene was purchased from Alfa Aesar. Sarcosine was bought from Sigma Aldrich and dried in vacuum before NCA synthesis. N-tert- butyloxycarbonyl (BOC)-ethylenediamine and A^f, A -di isopropyl ethylamine (DIPEA) were purchased from Sigma Aldrich, fractionally distilled and stored at -78 °C and -20 C, respectively. Oleic acid coated iron oxide nanoparticles were obtained from Sanofi-Aventis Deutschland GmbH, as well as from Ocean Nanotech, San Diego, USA. D,L-Lipoic and was bought from TCI Europe. Pentafluorophenyl trifluoroacetate, tris(2-carboxyethyl)phosphine (TCEP HCl) and acetic acid anhydride were obtained from Sigma Aldrich and used without further purification. Cyanine 5 NHS Ester was obtained from Lumiprobe GmbH. Animals

10 female C57B1/6 mice, aged 6 to 8 weeks, were housed in specific pathogen-free conditions under constant light-dark cycle and maintained on a standard mouse diet. Experimentation was performed at the DKFZ animal facilities, in accordance with institutional guidelines, and were approved by the Regierungsprasidium Karlsruhe, Germany, under permit number G214/19. Mice were anaesthetized by intrap eritoneal injection of 100 pg/g ketamine and 14 pg/g xylazine and intratracheally instilled with ION-CCPM (10 mg/kg of iron to body weight) or PBS in a final volume of 50 pL.

Instruments

'H, ¹⁹F and ¹³C NMR spectra were recorded on a Bruker Avance II 400 at room temperature at a frequency of 400, 376 and 100MHz and on a Bruker Avance III HD 300 at room temperature at a frequency of 300, 282 and 75 MHz, respectively. DOSY spectra were recorded on a Bruker Avance III HD 400 (400 MHz). Calibration of the spectra was achieved using the solvent signals (Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62 (21), 7512-7515). NMR spectra were analyzed with MestReNova version 12.0.0 from Mestrelab Research. Degrees of polymerization (An) by 'H NMR were calculated comparing the integral of the initiator peak and the integrals of the protons for pSar and pCys(SC>2Et), respectively. Attenuated total reflectance fourier transformed infrared (ATR-FTIR) spectroscopy was performed on a FT/IR-4100 (JASCO) with an ATR sampling accessory (MIRacle, Pike Technologies). IR spectra were analyzed using Spectra Manager 2.0 (JASCO) for integration. NCA polymerization was monitored by FT-IR spectroscopy. Polymerization was judged to be completed when NCA associated carbonyl peaks at 1853 and 1786 cm ¹ had vanished. UV-Vis spectra were recorded using a Jasco V-630 spectrophotometer (1 cm ^c 1 cm quartz cell).

Analytical gel permeation chromatography (GPC) was performed using HFIP as eluent, which contained 3 gL ¹ of potassium trifluoroacetate (KOTFA) at a flow rate of 0.8 mL min ¹ at 40°C. GPC columns were packed with modified silica (PFG-columns), particle size 7 pm, porosity 100 A and 1000 A, respectively, purchased from Polymer Standards Service GmbH. Poly(methyl methacrylate) standards (Polymer Standards Service GmbH) and pSar standards were used for calibration and toluene was used as the internal standard (Weber, B.; Birke, A.; Fischer, K.; Schmidt, M.; Barz, M. Macromolecules 2018, 51 (7), 2653-2661). A refractive index detector (G1362A RID, JASCO) and a UV detector (l = 230 nm, UV-2075+, JASCO) were used for polymer detection and analysis was performed using PSS WinGPC from PSS Polymer Standard Service GmbH.

Melting points of NCAs were determined with a Mettler FP62 melting point apparatus at a heating rate of 2.5 _C/min. Field desorption mass spectrometry (FD-MS) was performed on a FD Finnigan MAT90 spectrometer and electrospray ionization mass spectrometry (ESI- MS) was performed on a Micromass Q-TOF-Ultima spectrometer. Centrifugation was carried out in a Thermo Scientific Heraeus Multifuge 1 and in a Thermo Scientific Heraeus MFresco. Partitition coefficients (logP values) were calculated using MarvinSketch version 16.7.18.0 (ChemAxon Ltd.).

Thermogravimetric analysis (TGA) was performed on a Pyris 6 thermogravimetric analyzer (Perkin Elmer Inc.) using Pyris software. Analysis of lyophilized particle samples was performed in pure oxygen atmosphere at a heating rate of 10°C/minute from 50 to 800 °C. The mass concentration of iron was calculated from the residual iron oxide.

Atomic force microscopy (AFM) was measured on mica using a Cypher TM AFM (Asylum Research) using tapping mode at a scan rate of 1 Hz. Samples were prepared by drop-casting of a particle solution (b = 50 mg-L ¹ in MilliQ water) onto freshly cleaned mica. The sample was dried overnight at room temperature. Images were evaluated using Gwyddion 2.49.

Transmission electron microscopy (TEM) was performed on a FEI Tecnai G2 Spirit microscope equipped with a Gatan US 1000 2k x 2k CCD camera and LaB₆ cathode operated at 120 kV. Images were recorded using freshly glow discharged carbon coated copper grids (CF300-Cu, 300 mesh). For non-stained samples, 5 pL nanoparticle solution (b = 50 mg-L ¹ in MilliQ water) was drop-coated on the TEM grid surface and removed with a filter paper after 1 min. For negatively stained samples, 5 pL nanoparticle solution (b = 50 mg-L ¹ in MilliQ water) was drop-coated on the TEM grid, removed with a filter paper after 1 minute. Next, 5 pL uranyl acetate solution (2 wt.% in ethanol) were added and removed after 15 s incubation time. All sample-deposited grids were air-dried overnight before measurement. Software ImageJ 1.52h (National Institutes of Health, USA) was used for image evaluation.

Single-angle dynamic light scattering (DLS) measurements were performed with a ZetaSizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK) equipped with a He-Ne laser (l=632.8 nm) as the incident beam. All measurements were performed at 25°C and a detection angle of 173° unless stated otherwise. Disposable polystyrene or PMMA cuvettes (VWR, Darmstadt, Germany) were used for single-angle DLS measurements. Disposable folded capillary cells (Malvern Instruments Ltd., Worcestershire, UK) were employed for zeta potential measurements. Zeta potential measurements were conducted in solutions containing 3 mM sodium chloride. Cumulant size, polydispersity index (PDI), and size distribution (intensity weighted) histograms were calculated based on the autocorrelation function of the samples, with automated position and attenuator adjustment at multiple scans (typically 3 x 10-15 scans). For aggregation experiments the derived countrate was used.

For multi-angle DLS cylindrical quartz cuvettes (Hellma, Miihlheim, Germany) were cleaned by dust-free distilled acetone and transferred to a dust free flow box. Light scattering measurements were performed on an ALV spectrometer consisting of a goniometer and an ALV-5004 multiple-tau full-digital correlator (320 channels) which allows measurements over an angular range from 30° to 150°. A He-Ne Laser (l=632.8 nm) was used as light source. The correlation functions of the particles were fitted using a sum of two exponentials. The z-average diffusion coefficient D_z was calculated by extrapolating D_app for q = 0. By formal application of Stokes law, the inverse z-average hydrodynamic radius is R_h= (/Of¹)/¹ was determined. To investigate the aggregation behavior of the particles in human plasma, undiluted citrate plasma was filtered through a Millex GS 0.2 pm filter. The particle solutions were filtered through 0.45 pm pore size Millex LCR filters. The following mixtures were prepared from initial particle solutions in PBS (b = 1 g-L '): PBS/particle solution 9:1 (b = 0.1 g-L ¹), plasma/PBS 9:1 and plasma/particle solution 9:1 (b = 0.1 g-L ¹). The cuvettes were incubated for 30 min at room temperature before measurement at T = 20°C. Data analysis was performed according to a procedure reported by Rausch et al. (Rausch, K.; Reuter, A.; Fischer, K.; Schmidt, M. Biomacromolecules 2010, 11 (11), 2836-2839). The correlation functions of plasma were fitted with a triexponential decay function, while the particles were fitted using a sum of two exponentials. Mixtures were fitted using a sum of both exponential decay functions with or without an additional aggregate term.

Preparation of ION-loaded micelles

Oleic acid-coated iron oxide nanoparticles (IONs) (b = 5.8 g L ¹, 9.0 mL) were precipitated into 40 mL of ethanol, sonicated for 15 minutes and sedimented (4500 rpm, 15 min, 20°C). The pellet was resuspended in 5.0 mL of chloroform, sonicated for 30 minutes, precipitated in 45 mL of ethanol, and sedimented (4500 rpm, 15 min, 20°C) to remove excess oleic acid. IONs were resuspended in 20 mL of chloroform and a solution of pSar-b- pCys(S0₂Et) in DMSO/CHCh (1:2) (b = 5.0 g L ¹, 10 mL) was added dropwise. The resulting clear brown solution was placed in a dialysis bag (MWCO 3.5 kDa) and dialyzed against CHCh, followed by dialysis against DMSO. The solution was diluted with DMSO by factor 2 and dialyzed against MilliQ water to obtain ION-loaded polymeric micelles. The obtained ION-loaded micelles were filtered through a PVDF 0.45 pm filter and concentrated to a total volume of 8.0 mL by spin filtration (Amicon Ultra-15, MWCO 3.0 kDa, 4500 rpm, 20°C).

Preparation of ION-loaded core cross-linked polymeric micelles (ION-CCPMs)

For core cross-linking, D,L-lipoic acid (8.0 mg, 39.1 mmol, 0.5 eq. per pCys(S0₂Et) repeating unit) was dissolved in DMSO (5.0 g L ¹) and treated with tris(2- carboxyethyl)phosphine hydrochloride (11.2 mg, 39.1 mmol, 50 g L ¹ in MilliQ water) for 18 h yielding dihydro lipoic acid. This solution of dihydro lipoic acid was subsequently added to the ION-loaded micelle solution and the reaction mixture was placed on a benchtop shaker for 24 h. Subsequently, excess cross-linker and residual oleic acid were removed by dialysis (MWCO 3.5 kDa) against DMSO/MilliQ water mixtures (1:1) followed by dialysis against MilliQ water yielding a clear light brown solution of ION-loaded core cross-linked micelles (ION-CCPMs).

Dye conjugation (ION-CCPM^Cy5)

The ION-CCPM solution was adjusted to pH 7.4 using 1 M sodium hydrogen carbonate solution, Cy5-NHS ester (540 pg, 0.3 eq. per polymer, 25 g L ¹ in DMSO) was added and the solution was stirred at room temperature for 72 h. Upon addition of the blue dye solution, the particle solution turned dark green immediately. Excess dye was removed by repetitive extraction with dichloromethane, followed by dialysis against ethanol/MilliQ water mixtures (1:1) and MilliQ water (MWCO 6-8 kDa). Cy5-labelled SPION-loaded core cross- linked polymeric micelles (ION-CCPM⁰⁵'⁵) were concentrated to a total volume of 8.53 mL by spin filtration (Amicon Ultra-15, MWCO 100 kDa, 3000 rpm, 20°C), yielding 23 mg of SPION-CCPM^Cy5 (overall yield 23%).

Isolation of bone marrow derived macrophage (BMDM)

BMDMs were differentiated in vitro from bone marrow stem cell progenitors for one week using RPMI medium supplemented with 10 ng/ml M-CSF (M9170, Sigma- Aldrich), 10% FBS and 1% Penicillin/Streptomycin (Gibco) as described in Guida, C., Altamura, S., Klein, F.A., Galy, B., Boutros, M., Ulmer, A.J., Hentze, M.W., and Muckenthaler, M.U. (2015). A novel inflammatory pathway mediating rapid hepci din-independent hypoferremia. 725.).BMDMs were co-treated with 100 ng/ml LPS to obtain Ml macrophages. For each independent experiment, BMDMs were prepared from three different mice. Microscopy

BMDMs were plated on 13mm glass coverslips in a concentration of 3.5 x 10⁵ cells/slip. After incubation or treatment, cells were wash 3X with PBS and fixed with 4% paraformaldehyde for 15 minutes. Cells were then washed 3X with PBS and blocked with 2.5% milk in PBS-T (0.1% Tween) solution for 30 minutes on an orbital shaker. Slips were then washed 3X with PBS-T and incubated with primary antibody overnight at 4°C or 1 hour at room temperature. Primary antibody, Ibal, was diluted in 2.5% milk PBS-T. After washing with PBS-T 3X, slips were incubated with secondary antibody for 1 hour at room temperature. Secondary antibody was diluted in 2.5% milk PBS-T. Slips were then washed with PBS and mounted using Sigma Anti-Fade Gold mounting medium with DAPI. Samples were visualized and imaged at the Nikon Center, Heidelberg using a Ni-E confocal microscope. Images were analysed using ImageJ along with a written macro for intracellular quantification of the Cy5+ signal. Images were compiled using Adobe Photoshop and Illustrator.

Flow Cytometry

BMDMs were incubated with Fc-g receptor blocking solution and stained with anti mouse CD206-FITC, CD86-PE, MHC II-PeCy5, 7AAD (BioLegend, California, USA) and CD38-FITC (BD Biosciences). Data were acquired by a FACS Fortessa (BD, Biosciences) or Cytotek Aurora flow cytometer and analysis was performed using the FlowJo Software (Tree Star Inc) at the European Molecular Biological Laboratory (EMBL) Flow Cytometry Core Facility. The expression of surface markers is shown as Relative Fluorescence Units (RFU), whereby the geometric median fluorescence intensity (MFI) of the cells stained with the isotype-matched antibody was subtracted from the MFI of those stained with the specific antibody and normalized to the control (NT).

Cytotoxicity

BMDM viability was quantified using CytoTox96 kit from Promega. Briefly, cells were plated in a black side/black bottom 96 well plate at a concentration of 10,000 cells in 100 pL/well 24 hours before start of experiment. To measure LDH release into the supernatant, plate was centrifuged at 500 G for 10 mins to sediment cells and 100 pL was taken off each well and transferred to a new 96 well plate. 50 uL of substrate was added and plate was incubated for 30 minutes at room temperature in the dark. After 30 minutes, 20 pL stop solution was added to each well and 490 nm signal was measured on a spectrofluorimeter (SpectraMax, Molecular Devices). Viability was calculated by subtracting the media blank from experimental values, normalized to the control (NT). To measure redox capacity, after incubation times with conditions, 10 pL of Celltiter Blue was added to each well and plate was incubated at 37°C for 4 hours. Absorbance was then measured at 520 nm and all values were subtracted from the media blank control and normalized to the control (NT).

Quantitative Real-Time Polymerase Chain Reaction Analysis

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). 0.5 to 1 pg of total RNA was reverse transcribed by using RevertAid H Minus reverse transcriptase (Thermo Scientific), random primers (Invitrogen) and dNTPs (ThermoScientific). qRT-PCR was performed on a Step One Plus Real Time PCR System (Applied Biosystems, California, USA). Primers and probes were designed using the ProbeFinder software (www.roche- applied-science.com).

Measurement of intracellular ROS accumulation

Accumulation of ROS in BMDM cells was assessed by using the oxidant- sensitive fluorescent dye CELLROX™ Green and CELLROX™ Orange (Life Technologies). Upon crossing the cellular membrane, the non-fluorescent CELLROX™ probe undergoes deacetylation by intracellular esterases producing a highly green fluorescent following oxidation by intracellular ROS. BMDMs were maintained untreated or treated for 4 or 18 hours with ION-CCPMs, CCPMs, Lipopolysaccharide (LPS), ferric ammonium citrate (FAC), and heme. Then 2.5 mM of CELLROX™ Green or Orange was incubated for 30 minutes at 37 °C under 5% CO2 atmosphere. Cells were then washed twice with HBSS, and fluorescence intensity was measured using FACS. Fluorescence intensity is represented as median fluorescent intensity (MFI).

Protein Extraction and Western Blotting

Protein extracts were prepared as previously reported and protein concentration was determined using the Bio-Rad protein assay system (Bio-Rad, Miinchen, Germany). 50 pg of total protein extracts were separated by 8-12% SDS-PAGE and analyzed by Western blotting using antibodies against HO-1 (Stressgen, Victoria, Canada), TfRl (Invitrogen/Life Tech) and actin (Santa Cruz). Densitometric analysis is reported in Arbitrary Unit (AU), as ratio to the untreated (NT) sample (AU=1). Peris’ Prussian blue staining

BMDMs were plated on 13 mm glass coverslips in a concentration of 3.5 x 10⁵ cells/slip. After incubation or treatment, cells were washed 3X with PBS and fixed with 4% paraformaldehyde for 15 minutes. Cells were then washed 3X with PBS and stained with Accustain Iron Stain No. HT20 (Sigma- Aldrich) following manufacturer’s instructions.

Buffy Coat preparation

Human monocytes were isolated from commercially available buffy coats (DRK- Blutspendedienst Baden-Wurttemberg-Hessen, Frankfurt, Germany) using Ficoll-Hypaque gradients (PAA Laboratories). Monocytes were differentiated into primary human macrophages with RPMI 1640 containing 5% AB-positive human serum (DRK- Blutspendedienst) for 7 days and achieved approximately 80% confluence. 24 hours prior to stimulation, cells were serum starved.

Example 1:

Nanoparticle Preparation and Characterization

Reactive amphiphilic pSar-b-pCys(S0₂Et) block copolypept(o)ides have been synthesized by nucleophilic ring-opening NCA polymerization. Block copolymer synthesis yielded 2.9 g of P2 and 2.3g of P3, respectively. The reaction scheme is shown in Fig. 1. The following table 2 shows the characterization of pSar_n-6/ocA-pCys(S02Et)m copolymers.

Table 1 polymer X_n pSar^[a] X_n wt.% M_n ^[cl D ^[c] pCys(S0₂Et)^[bl Cys(S0₂Et)

P1 225 33 28.7 31150 2.64

P2 200 17 18.9 31700 1.25

P3 170 29 31.9 35100 7.06

[a] Obtained by HFIP-GPC, relative to pSar standards [b] Acquired by ¹H-NMR. [c] Obtained by HFIP-GPC relative to PMMA standards.

Single-angle DLS of pSar-b-pCys(S02Et) block copolymers (PI - P3) in DMSO is shown in Figure 14. ¾ DOSY NMR spectra of PI (pSar225-block-pCys(S02Et)33) in DMSO- d6 is shown in Figure 15, of P2 (pSar₂oo-block-pCys(S0₂Et)i₇) in Figure 16, and of P3 (pSari7o-block-pCys(S02Et)2₉) in Figure 17.

As illustrated in Figure 2, ION-CCPMs were prepared by self-assembly of commercially available IONs in the presence of pSar-b-pCys(SC>2Et) block copolymers. To obtain well-defined ION-loaded micelles, briefly, block co-polymers were dissolved in a mixture of DMSO and chloroform (1:2), added to a dispersion of oleic-acid-coated IONs and dialyzed against chloroform, DMSO and water. Upon solvent exchange, micelles were core cross-linked with dihydro lipoic acid, resulting in the formation of bio-reversible disulphide bonds in the core compartment (ION-CCPM). To allow for biological evaluation, the fluorescent dye Cy5 was conjugated to the primary amine end group (ION-CCPM^Cy5). Upon addition of Cy5 (blue), the orange solution of ION-CCPM immediately turned dark green (Figure 3A). Removal of unconjugated dye could be done by repetitive extractions with dichloromethane (Figure 3B).

According to single-angle dynamic light scattering (DLS), co-self-assembly of oleic acid-coated IONs and P3 yielded structures with D_h = 71 nm (Figure 3C). After core cross- linking with dihydro lipoic acid, dye conjugation and particle purification the hydrodynamic diameter slightly increased to D_h = 82 nm. The size distribution of ION-CCPM⁰⁵'⁵ remained unchanged when particles were lyophilized and re-dispersed in water at the identical concentration. Based on our experience, this can be considered an indication of successful micelle cross-linking and particle purification.

Further analysis of ION-CCPM^Cy5 by atomic force microscopy (AFM) revealed spherical structures with sizes below 100 nm (Figure 3D), which is congruent with DLS and fluorescence correlation spectroscopy (FCS) data (Table 2).

Table 2. Summary of the prepared particles. [A]: Derived from residual weight by thermogravimetric analysis. [B]: Determined by single-angle dynamic light scattering at an angle of 173°. [C]: Determined by fluorescence correlation spectroscopy.

CCPMs^⁵ PI, pSar₂₂₅-ft-pCys(S0₂Et)3i - 20.9 49 0.13 47 -4.2

ION/polymer co-self-assembly mimics a template-assisted process which accounts for the formation of spherical structures. Complementary to AFM, transmission electron microscopy (TEM) was used to elucidate the encapsulated iron oxide nanoparticles. As shown in Figure 3E, iron oxide nanoparticles were found to be organized in patterns of local clusters with total dimensions below 50 nm containing multiple cores each. The single cores showed diameters of 6 to 10 nm. In contrast, oleic acid-coated IONs were found randomly arranged, as processed from hexane dispersions. Since the polymer shell could not be visualized due to large contrast discrepancies, the observed local clustering emphasizes successful encapsulation of iron oxide nanoparticles into core cross-linked polymeric micelles.

ION-CCPMs show colloidal stability and stimuli-responsive degradation

To confirm stable cross-linking, ION-CCPM^Cy5 were incubated in hexafluoroisopropanol (HFIP) for at least 1 h before analysis by gel permeation chromatography (GPC) in HFIP (Figure 4C). The signal at 12.5 mL combined with the absence of signals at elution volumes of 17 and 20 mL verified successful particle stabilization, as well as elimination of unconjugated polymer and dye. In addition, the absence of unconjugated dye was verified by FCS.

ION-CCPM^Cy5 exhibit low negative z-potentials of -5.1 and -5.5 mV, accounting for efficient shielding of the iron oxide surface charge by the polysarcosine corona (Figure 4D) and is comparable to unloaded particles.

The preparation and characterization of ION-CCPMs are mostly described from a materials science perspective indicating particle integrity, while referring to the concepts of core cross-linking and iron oxide encapsulation and functionalization. However, for systemic administration in vivo , unspecific interaction with plasma proteins is considered a severe obstacle for prolonged circulation time and site-specific (drug) delivery. Thus, according to the procedure established by Rausch et ak, DLS was performed in human blood plasma (K. Rausch, A. Reuter, K. Fischer and M. Schmidt, Biomacromolecules, 2010, 11, 2836-2839; K. Fischer and M. Schmidt, Biomaterials, 2016, 98, 79-91). This technique can be utilized to monitor nanoparticle-induced aggregation of plasma components with high sensitivity (P. Heller, D. Hobemik, U. Lachelt, M. Schinnerer, B. Weber, M. Schmidt, E. Wagner, M. Bros and M. Barz, J. Control. Release, 2017, 258, 146-160). As shown Figure 4E, no aggregation was detected after incubation in human blood plasma with ION-CCPM^Cy5 at a concentration of 100 mgL ¹ .

The stimuli-responsive behavior of disulfide cross-linked ION-CCPMs was evaluated by DLS in carbonate buffer (pH 7.4) at extra- and intracellularly relevant concentrations of glutathione (GSH). At extracellular GSH levels (10 mM) the derived count rate remains constant, while a decrease was observed at intracellular GSH levels (10 mM), thus indicating particle degradation (see Figure 17). Interestingly, when the same experiment was conducted in phosphate buffer (pH 7.4), precipitation of iron oxide/phosphate could be observed for ION-CCPMs treated with GSH concentrations above 10 mM (see Figure 18), which exemplifies the accessibility of the encapsulated iron upon redox-dependent particle degradation.

Example 2:

ION-CCPMs and CCPMs are preferentially taken up by macrophages

In this example it was tested whether the iron containing polymer shells (ION- CCPMs) or the polymer shells (CCPMs) used as controls particles are taken up by macrophages. Primary bone marrow derived macrophages (BMDMs) were incubated with increasing concentrations of ION-CCPMs and CCPMs for 24 hours. Amount of ION-CCPMs added to cells was calculated based on concentration of iron contained the core, at 1, 4 and 20 mM of iron. The amount of CCPMs added to cells was calculated to match the mass of CCPMs contained within ION-CCPMs at each concentration. Internalization of nanoparticles was measured by intracellular fluorescent intensity using Fluorescence-activated cell sorting (FACS) and fluorescence microscopy. FACS analysis showed that at a 1, 4, and 20 mM concentration, ION-CCPM particles were internalized more efficiently than CCPMs, consistent with microscopy observations (Figure 5). At 1 hr time point, uptake of both particles was observed, indicating the rapid rate of uptake by macrophages (Figure 6). Data reported as n ± SEM and normalized to either background (Figure 5A) or non-treated condition (NT) (Figure 5C). n = 3 independent experiments, Figure 5B and Figure 6 show representative images. One-way ANOVA: *p < 0.05, ** ? < 0.01, ***p < 0.001.

Example 3:

ION-CCPMs do not cause cytotoxicity in BMDMs

It was investigated whether cellular uptake of ION-CCPMs or CCPMs affect cell viability. Figure 7 shows that ION-CCPMs stimulate BMDMs. Macrophages were incubated with 20 mM ION-CCPMs, CCPMs, or ferric ammonium citrate (FAC). In Figure 7A, supernatants of cultures were used to measured lactate dehydrogenase (LDH) quantities at

490 nm after adding CytoTox 96© substrate (Promega). Values are represented as a percentage of 100% viable control at each time point. In Figure 7B, after experimental incubation time, cells were incubated with CellTiter-Blue (Promega) for 4 hours and fluorescence was measured at 590 nm. Values are represented as fold change of 100% cell death value. Data reported as n ± SEM. n = 3 independent experiments. One-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, * indicates comparison to NT. These data suggest that both cysteine and iron can act together to stimulate the metabolic activity of macrophages.

Example 4:

ION-CCPMs are catabolized and release metabolicallv active iron in BMDMs

It was investigated whether iron contained in ION-CCPMs is metabolically active upon nanoparticle breakdown. To this end, gene expression of iron regulatory genes was analyzed. At 4 hours, a decreased mRNA expression of transferrin receptor 1 (TFR1) was observed, a molecular marker of high intracellular iron content (Figure 8A), demonstrating that iron released from ION-CCPMs is metabolically active. Similar results were obtained when BMDMs were treated with 20 mM FAC. A recent study demonstrated that cysteine- related toxicity can occur due to limiting iron availability and inhibition of mitochondrial activity (CE. Hughes, TK. Coody, M. Jeong, JA. Berg, DR. Winge, AL. Hughes, Cell, 2020, 18, 296-310). Therefore, elevated TfRl mRNA levels in CCPM treated cells may be explained by cysteine related toxicity. In addition, CCPMs induce HO-1 protein expression (Figure 8A), an intracellular stress marker. In addition, BMDMs treated with ION-CCPMs express high mRNA levels of the iron exporter Ferroportin (Fpnl) (Figure 8B), possibly as a safety mechanism to prevent toxic iron overload. Treatment of BMDMs with the intracellular iron chelator deferiprone (DFI) together with ION-CCPMs reverted the increase of Fpnl mRNA levels to those observed in BMDMs treated with DFI only. Increased Fpnl expression is likely caused by free cytoplasmic iron to counteract oxidative stress in ION-CCPM treated BMDMs. Consistently, 4 hours after ION-CCPM treatment, high cytoplasmic and normal nuclear and mitochondrial ROS levels were observed (Figure 8C), suggesting that free cytoplasmic iron is released soon after nanoparticle internalization and breakdown. Interestingly, 18 hours after ION-CCPM treatment, ROS detection shifts from the cytoplasm to the nucleus and mitochondria (Figure 8D), where results are comparable to those in FAC and iron dextran treated cells. In addition, at the 24h time point, BMDMs appear intact and iron stores are detectable by Peris’ prussian blue stain (Figure 8D), together with a reduction in Fpnl mRNA levels (Figure 8E). Importantly, CCPMs do not increase ROS levels in BMDM, additionally indicating that iron triggers ROS production (Figure 8C). The strongest signal for iron is detected at the 24-hour time point (Figure 8D). This suggests that ION- CCPMs are continuously degraded with slow kinetics over an extended time period and that BMDMs can safely handle the internalized particles, avoiding necrosis or other adverse effects. CCPMs and ION-CCPMs thus induce little adverse cellular effects and present a good safety profile.

The phenotype of macrophages exposed to heme or non-transferrin bound iron shifts towards an inflammatory state, hallmarked by increased levels of inflammatory cytokines, such interleukin (IL)-l a/b, IL-6, and tumor necrosis factor (TNF)a, as well as elevated expression of pro-inflammatory cell surface proteins, such as Cluster of Differentiation (CD) 86, CD80 and Class II major histocompatibility complex molecules (MHC II). The pro- inflammatory surface markers and cytokine levels in BMDMs exposed to 100 ng/mL lipopolysaccharide (LPS), 20 mM of an iron source (heme), ION-CCPMs or CCPMs were therefore investigated. It is shown that BMDMs treated with ION-CCPMs increase the expression of CD86, CD38, MHC II and CD80, similar to LPS stimulated cells (Figure 9 A). In addition, inflammatory cytokines, such as TNFa, iNOS, CXCL10, IL6, and IL l b, were activated in cells treated with ION-CCPMs (Figure 9B). Furthermore, expression of the mannose receptor, CD206, an indicator of anti-inflammatory phenotypic activation, was significantly lower in BMDMs exposed to ION-CCPMs compared to those treated with CCPMs (Figure 9C). Importantly, the inflammatory response to ION-CCPMS was not restricted to mouse BMDMs but similarly occurs in human macrophages (Figure 10). Finally, it was tested whether individual components of the nanoparticle, such as L-cysteine, ethylated cysteine dimers, or cysteine homodimers, with or without iron, induce an inflammatory response. Addition of any variation of cysteine did not induce inflammatory markers, though cysteine together with an iron source (FAC or heme) induced expression of CD86, albeit to a lower extent than intact ION-CCPMs (Figure 11). The chemical nature of the nanoparticles together with the release of iron is thus considered responsible for the observed inflammatory responses. Taken together, ION-CCPMs are a potent immunostimulatory agent in murine and human macrophages, demonstrating their potential as a putative immunotherapeutic.

Example 5:

Macrophage stimulation by ION-CCPMs resembles signaling induced by reactive iron or heme

Already 4h after ION-CCPM treatment of BMDM, iron responses are induced as indicated by alterations in Fpnl, TFR1 and ROS levels (Figure 8). At the 18-hour time point a decrease in Fpnl levels was observed (Figure 8E), even though cells are iron-loaded (Figure 8D). Increased unbound iron causes ROS production (Figure 8C) and consequently, antioxidant signaling is induced. Expression of antioxidant genes is induced by the transcription factor NF-E2 p45-related factor 2 (Nfr2). Here, the expression of three Nrf2 target genes, NAD(P)H dehydrogenase (quinone) 1 (Nqol), Glutathione S-Transf erase Mu 1 (Gstml) and Suppressor of cytokine signaling 3 (Socs3) we examined. It was found that these are significantly increased (Figure 12A, B, C). This suggests that ION-CCPMs may induce sterile inflammation in macrophages. The Nrf2 response is further activated by heme treatment of BMDMs and is different from those responses induced by LPS stimulation. Despite this, BMDMs treated with ION-CCPMs increase Soc3 mRNA levels similar to LPS signaling (Figure 12C) but fail to increase arginase mRNA expression (Figure 12D). Without wishing to be bound by any theory, it is assumed that a cross-talk of signaling pathways exists that responds to iron or inflammatory patterns, such as LPS, that are operational upon ION CCPM treatment.

Example 6:

Intratracheal instillation of ION-CCPMs polarizes lung macrophages and stimulates innate immune lung cells

ION-CCPMs induce inflammation in vivo. Female mice, aged 6 to 8 weeks, were intratracheally instilled with phosphate-buffered saline (PBS) or ION-CCPMs. At 4, 24, 48 and 96 h post-administration, mice were sacrificed and evaluated for immune cell recruitment, phenotyping of immune cells and iron content in the lungs.

As the lungs are densely populated with macrophages, ION-CCPMs can be applied non-invasively to macrophages while at the same time reducing off-target immune activation in other organs. Therefore, intratracheal administration was a preferred method of application. At 24 h after instillation, non-heme iron content increased approximately threefold in the lungs of ION-CCPMs administered mice compared to PBS administered mice (Figure 19). At 96 h, a fivefold increase in iron content of the lungs was observed indicating that iron is released from ION-CCPMs over time and is absorbed into the lung tissue. Other organs, such as the liver, were surveyed for changes in non-heme iron content and showed little signs of increased iron deposition. This indicates that iron of ION-CCPMs remains at the site of application rather than distributing systemically. This is also shown by the lack of alterations in hematological parameters measured in both groups of mice (Figure 20).

Flow cytometry analysis of the lung cells indicates that ION-CCPMs stimulate an acute immune response within 4 h, which lasts up to 96 h (Figure 21). Samples were prepared by generating a single cell suspension using the Lung dissociation kit from Miltenyi. ION- CCPM⁺ cells were detected as early as 4 h after administration in interstitial macrophages (IM) (Figure 21 A). After 24 h, other innate immune cells were observed to accumulate ION- CCPM⁺ fluorescence signal, including neutrophils, eosinophils and dendritic cells, with neutrophils showing the brightest signal out of all cell types. The brightness in signal in neutrophils also corresponds to an extensive recruitment of neutrophils in the lung tissue at 24 h (Figure 2 IB). After 48 h, the brightest signal intensity of ION-CCPMs was detected in dendritic cells indicating the dynamics of ION-CCPM degradation upon internalization in innate immune cells.

Evaluation of the inflammatory response in the lungs of mice upon administration of either PBS or ION-CCPMs demonstrates that inflammatory signaling was initiated as early as 4 h in macrophages (Figure 22). This is shown by an increase in the cell surface marker levels of CD80 (a known inflammatory protein) and a decrease in anti-inflammatory cell surface markers such as C-Mer proto-oncogene tyrosine kinase (MerTK, a protein expressed under conditions when inflammation resolves) found at 4 h on Alveolar macrophages (AM). IM were also responsive to ION-CCPMs, showing reduced CD71 levels at the 24 h time point indicating a time-dependent intracellular degradation of ION-CCPMs triggering a well-known response to iron accumulation. This coincides with increased accumulation of ION-CCPMs in IMs after 24 h (Figure 21). mRNA from lung tissue was extracted by using the Trizol method for RNA preparation. Samples were then used to prepare cDNA by undergoing RT-PCR. The inflammatory response in lung tissue was further substantiated by showing time-dependent mRNA expression of the pro-inflammatory cytokines 111/5, 116 and Tnftx, as well as of oxidative stress response proteins Ho-1 and Slc7all in fold-change over PBS (F.C. vs PBS) (Figure 23).

Example 7:

ION-CCPM polarized macrophages reduce cancer cell proliferation and induce oxidative stress

Lewis lung carcinoma (LLC) cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye prior to culturing with bone marrow derived macrophages (BMDMs). Macrophages and Lewis lung carcinoma cells were co-cultured over a 72 h period. Viability, rate of division and intracellular LLC signal intensity in macrophages were sampled at 6, 12, 24, 48 and 72 h after the addition of ION-CCPMs or CCPMs. A reduced number of viable LLC cells was found in cultures treated with ION-CCPMs compared to CCPMs or non- treated cultures starting at 24 h (Figure 24A) while macrophages maintained a consistent cell population over time (Figure 24B). Starting from 12 h, LLC cells from cultures treated with ION-CCPMs show a diminished division rate compared to the CCPM treated LLC cells (Figure 24C). Macrophages were found to have the brightest intensity of LLC derived fluorescence signal at 24 h upon ION-CCPM treatment compared to controls (Figure 24D), indicating an increase in phagocytic activity stimulated by ION-CCPMs. Macrophages accumulated the brightest ION-CCPM signal compared to LLC cells with increasing intensity over time whereas CCPMs were accumulated more abundantly in LLC cells rather than macrophages (Figure 24E).

Gene expression was evaluated in LLC cells and macrophages after co-culturing and ION-CCPM or CCPM treatment for 24 or 48 h. Upon ION-CCPM treatment, LLCs upregulated the oxidative stress gene Nqol when co-cultured with macrophages and not with CCPMs or when cultured independently (Figure 24F). Macrophages, either cultured with LLC cells or independently, significantly upregulated the expression of NOS2 , which encodes for the iNOS enzyme responsible for the secretion of nitric oxide species (Figure 24G). An increase of the NOS2 gene in macrophages upon CCPM treatment was found only at 48 h when cultured alone.

Example 8:

ION-CCPMs alter the immune landscape in lung tumor bearing mice by increasing iron within the tumor microenvironment and affecting tumor number 12 female Friend leukemia virus B (FVB) mice (6 to 8 weeks of age) were intratracheally instilled with an advenovirus harbouring the EML4-Alk⁺ transposon mutation (2e8 PFU) for inducing lung cancer. After six weeks, mice were treated with either ION- CCPMs (10 mg/kg iron), CCPMs, or PBS intratracheally in a volume of 50 pi. After two administrations, necropsy was performed and immune cell populations within lung tumors were evaluated by flow cytometry. ION-CCPM treated mice were found to have an increased number of CDl lb⁺ F4/80⁺ cells, indicative of macrophages, within lung tumors in comparison to both CCPM and non-treated mice (Figure 25 A). Lung tumors were evaluated for iron content by Peris’ Prussian blue iron stain and DAB enhanced staining. Lung tumors were found to accumulate iron in the tumor microenvironment in ION-CCPM injected mice (Figure 25B, black arrows).

Eight female mice, (6 to 8 weeks of age) were treated intratracheally with ION- CCPMs (50 mg/kg) or were left untreated, and analyzed two weeks after viral infection as described above. A reduced tumor burden (indicated as number of tumors identified per mouse) was observed in mice treated with ION-CCPMs compared to PBS mice (Figure 25C).

Claims

1. A core-shell particle comprising

(i) a core cross-linked polymeric micelle (CCPM), and

2. The core-shell particle of claim 1, wherein the one or more IONs comprise Fe₂0₃ or Fe₃0₄ or a mixture thereof; and/or wherein the IONs have a diameter in the range of 5 to 20 nm.

3. The core-shell particle of claim 1 or 2, wherein the IONs are paramagnetic, preferably superparamagnetic.

4. The core-shell particle of any one of claims 1 to 3, wherein the one or more ION is coated with a small molecule surfactant, preferably wherein the small molecule surfactant is one or more fatty acid or monophosphoryl lipid, more preferably wherein the small molecule surfactant is one or more monounsaturated fatty acid, most preferably wherein the small molecule surfactant is oleic acid.

5. The core-shell particle of any one of claims 1 to 4, wherein the CCPM comprises a polymer comprising a thiol-reactive block consisting of between 1 and 1000 monomeric units of formula (C)

wherein n is 1 or 2;

R¹ is H, Ci-C4-alkyl, C2-C4-alkenyl or C2-C4-aklynyl;

(B)

wherein m is 1, 2, 3, 4, or 5;

R³ is independently selected from halogen, nitro, cyano and Ci-C4-alkanoyl;

R⁴ is selected from R^a, Ci-Ci₆-alkyl, R^a-Ci-Ci₆-alkyl, C2-Ci6-alkenyl, and C2- Ci₆-alkynyl, wherein the Ci-Ci₆-alkyl, C2-Ci6-alkenyl, and C2-Ci6-alkynyl are unsubstituted or carry 1, 2 or 3 substituents R^b, wherein R^b is independently selected from halogen, cyano, nitro, hydroxyl and thiol; wherein R^a is selected from

(i) phenyl;

(ii) 5- or 6-membered heteroaromatic monocyclic radicals having 1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N;

(iii) 8- to 10-membered heteroaromatic bicarbocyclic radicals having 1, 2, 3 or 4 heteroatoms as ring members which are independently selected from O, S and N; and

6. The core-shell particle of claim 5, wherein each R³ is independently selected from the group consisting of fluoro, bromo and chloro, preferably wherein m is 5 and R³ is chloro, or m is 1, 2 or 3 and R³ is fluoro, and/or

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7. The core-shell particle of any one of claims 1 to 6, further comprising at least one dye, wherein the at least one dye is preferably conjugated to the amine group of the amphiphilic copolymer.

8. A composition comprising a plurality of the core-shell particles according to any one of claims 1 to 7, optionally, further comprising a pharmaceutically acceptable carrier.

9. The core-shell particle of any of claims 1 to 7 or the composition of claim 8 for use in medicine.

10. The core-shell particle of any of claims 1 to 7 or the composition of claim 8 for use in immunotherapy or for use in treating (i) dysregulation of the immune system, (ii) cancer, or (iii) anemia.

11. A method of producing an iron oxide nanoparticle-loaded core cross-linked polymeric micelle, the method comprises the steps of:

(b) core cross-linking the cysteine moieties of the polymers; and

(c) optionally conjugating a dye to the micelles.

12. The method according to claim 11, wherein step (a) further comprises dialyzing the solution comprising the IONs and the amphiphilic reactive block copolymers against organic solvents and subsequently against water; and/or wherein step (b) further comprises dialyzing the solution comprising the core cross- linked polymeric micelles against organic solvents and subsequently against water.

13. The method according to claim 11 or 12, wherein the iron oxide nanoparticles in step (a) are coated with a small molecule surfactant, preferably wherein the small molecule surfactant is one or more fatty acid or monophosphoryl lipid, more preferably wherein the

3 small molecule surfactant is one or more monounsaturated fatty acid, most preferably wherein the small molecule surfactant is oleic acid.

14. The method according to any one of claims 10 to 13, wherein the CCPM comprises a plurality of amphiphilic polysarcosine-block-poly(S-alkylsulfonyl cysteine) copolymers and/or amphiphilic polysarcosine-block-poly(S-alkylsulfonyl homocysteine) copolymers, which are reacted with thiol-based cross-linkers.

15. An iron oxide nanoparticle-loaded core cross-linked polymeric micelle obtained by the method of any one of claims 11 to 14.

16. A method for modulating

(i) activity of immune cells, comprising administering the composition according to claim 8 to one or more immune cells, preferably wherein the immune cell is a macrophage, more preferably wherein activating the activity of the macrophage comprises inducing a pro- inflammatory response in the macrophage or inducing macrophage polarization; or

(ii) dendritic cell activity, comprising administering the composition according to claim 8 to one or more dendritic cells, preferably wherein activating dendritic cell activity comprises inducing a pro-inflammatory response in the one or more dendritic cells or inducing dendritic cell polarization; or

(iii) monocyte activity, comprising administering the composition according to claim 8 to one or more monocytes, preferably wherein activating monocyte activity comprises inducing a differentiation response in monocytes or inducing monocyte differentiation or maturation.

17. A method of treating dysregulation of the immune system in a patient in need thereof, the method comprising administering an effective amount of the composition according to claim 8 to the patient in need thereof.

18. The method according to claim 17, wherein the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition.

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19. A method of treating cancer in a patient in need thereof, the method comprising administering an effective amount of the composition according to claim 8 to the patient in need thereof.

20. The method according to claim 19, wherein the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition.

21. The method according to claim 19 or 20, wherein the cancer is lung cancer.

22. A method of treating anemia in a patient in need thereof, the method comprising administering an effective amount of the composition according to claim 8 to the patient in need thereof.

23. The method according to claim 22, wherein the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition.

24. A method of treating a nerve injury in a patient in need thereof, the method comprising administering an effective amount of the composition according to claim 8 to the patient in need thereof.

25. The method according to claim 24, wherein the administration of the composition to the patient in need thereof is intratracheal, via inhalation or intravenous injection of the composition.

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